This invention relates generally to methods and compounds for amplifying detectable signals in specific binding assays, particularly nucleic acid hybridization assays.
Nucleic acid hybridizations are commonly used in biochemical research and diagnostic assays. Generally a single stranded analyte nucleic acid is hybridized to labeled nucleic acid probe, and resulting nucleic acid duplexes are detected. Radioactive and nonradioactive labels have been used. Methods also have been developed to amplify the signal that is detected. For example, large comb-type branched polynucleotides, comprising a first oligonucleotide unit and branches including second oligonucleotide units, have been developed for signal amplification in nucleic acid detection assays. In this application, the branched polynucleotide is hybridized via the first oligonucleotide unit to single stranded analyte nucleic acid and then labeled oligonucleotide is hybridized to the branched polynucleotide via the second oligonucleotide units, as described in U.S. Pat. No. 5,710,264 to Chiron Corporation.
Dendrimers have been developed that are assembled by the sequential hybridization of single DNA strands. Pairwise hybridization of single strands produces monomers with a double stranded center and four single stranded xe2x80x9carmsxe2x80x9d. The monomers can be grown exponentially in sequential hybridization steps, to produce a macromolecule including terminal single stranded arms. The dendrimers may range in size from hundreds to millions of bases. The arms that are not to be hybridized to target can be hybridized to covalently labeled oligonucleotide. These dendrimers are commerically available from Polyprobe, Bala Cynwyd, Pa. Dendrimers for assaying nucleic acids are described in U.S. Pat. Nos. 5,175,270, 5,487,973, and 5,484,904, the disclosures of which are incorporated herein.
Avidin-biotin systems have been developed for use in a variety of detection assays. Methods for the detection and labeling of nucleic acids in biotin systems are described, for example, in xe2x80x9cNonradioactive Labeling and Detection Systemsxe2x80x9d, C. Kessler, Ed., Springer-Verlag, New York, 1992, pp. 70-99; and in xe2x80x9cMethods in Nonradioactive Detection,xe2x80x9d, G. Howard, Ed., Appleton and Lange, Norwalk, Conn., 1993, pp. 11-27 and 137-150.
Methods for the detection of nucleic acid sequences have suffered from drawbacks including background noise, time and labor requirements, lack of specificity and lack of sensitivity. It is an object of the invention to provide materials for the detection of polymers, particularly nucleic acids. It is a particular object of the invention to provide methods and compounds for amplifying labeling signals used in the detection of nucleic acid sequences in specific binding assays. It is a further object of the invention to provide methods and compounds which permit nucleic acid sequences to be detected specifically and rapidly with high sensitivity and high resolution.
Methods and compounds are provided for detecting target molecules using specific binding assays. Methods and compositions are provided that are useful in signal amplification in the detection of the target molecule.
In one embodiment, methods are provided for detecting a nucleic acid target, wherein the method comprises hybridizing a nucleic acid target, comprising a target nucleic acid sequence, to a nucleic acid probe, comprising a probe nucleic acid sequence, wherein the target comprises a binding ligand. The hydridized target is contacted with a receptor comprising multiple sites capable of binding the binding ligand to complex the receptor to the binding ligand, and the receptor is contacted with an amplification reagent, comprising a plurality of the binding ligands, to complex the amplification reagent to the receptor. The presence of the complexed amplification reagent then is detected, for example, by detecting the presence of a detectable label on at least one of the receptor and the amplification reagent.
Optionally, after complexing the amplification reagent to the receptor complexed to the hybridized target, the amplification reagent, comprising a plurality of the binding ligands, is contacted with labeled receptor molecules thereby to complex a plurality of labeled receptor molecules to the amplification reagent, and the labeled receptor molecules complexed to the amplification reagent are detected. This permits the detectable signal to be enhanced and more easily detected.
Labels which may be used include fluorescent labels, gold labels and enzyme labels. Exemplary fluorescent labels include fluorescein, rhodamine, resorufin, or a coumarin. In one preferred embodiment, the binding ligand is biotin and the receptor is avidin or streptavidin.
The amplification reagent may comprise a polymer, such as a poly(amino acid) or a polynucleotide. In one embodiment, the amplification reagent may be an antibody, such as an anti-receptor antibody capable of specifically binding the receptor.
In another embodiment, the amplification molecule may be a DNA matrix. The DNA matrix may include subunits of partially double stranded and partially single stranded DNA molecules. In one embodiment, the DNA matrix molecule includes a plurality of molecules of a first partially double stranded polynucleotide, the polynucleotide having a first molecule end, a second molecule end and a double stranded body portion intermediate of the first and second ends, wherein the first and second ends each comprise at least one of first and second arms consisting of a single strand of polynucleotide, and wherein the single strands are hybridizable with a predetermined nucleic acid sequence of nucleotides in a nucleic acid, and the first and second arms of each of said first and second ends are nonhybridizable with each other. The DNA matrix further includes a plurality of molecules of a second partially double stranded polynucleotide, each polynucleotide including a first molecule end, a second molecule end and a double stranded body portion intermediate the first and second ends, the first and second ends each having at least one of first and second arms consisting of a single strand of polynucleotide that is hybridizable with a predetermined nucleic acid sequence of nucleotides in a nucleic acid, wherein the first and second arms of each of said first and second ends are non-hybridizable with each other. In the DNA matrix, the plurality of molecules of the first polynucleotide and the second polynucleotide are joined together through annealing of one or more arms thereof, to form a matrix, wherein arms of the plurality of first and second polynucleotide molecules located on the outer surface of the matrix are hybridized to nucleic acids having the binding ligand attached thereto.
In one embodiment, the binding ligand is biotin and the receptor is avidin or streptavidin. For example, in the embodiment wherein the amplification reagent is an antibody the biotin may be covalently attached to the antibody. For example, the antibody may be an anti-streptavidin antibody comprising a plurality of biotin molecules covalently attached to the antibody. In an assay, after complexing of the antibody to a streptavidin receptor bound to the biotinylated target nucleic acid, the antibody may be contacted with labeled streptavidin, thereby to complex a plurality of labeled streptavidin molecules to the antibody, and the labeled streptavidin molecules complexed to the antibody then may be detected, thus providing signal amplification in the assay.
In the embodiment wherein the amplification reagent is a DNA matrix comprising single stranded DNA, a binding ligand consisting of biotin may be attached to the DNA matrix by hybridization of a plurality of biotinylated nucleic acids to single strands of the DNA matrix. In this embodiment, after complexing the DNA matrix to the receptor complexed to the hybridized target, the biotinylated DNA matrix is contacted with labeled streptavidin, thereby to complex a plurality of labeled streptavidin molecules to the biotins on the DNA matrix, and the labeled streptavidin molecules complexed to the DNA matrix are detected, thus enhancing the detectable signal.
In one preferred embodiment, the nucleic acid probe is immobilized on a surface. The surface may be, for example, Langmuir Blodgett film, glass, germanium, silicon, (poly)tetrafluorethylene, polystyrene, gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, and combinations thereof.
In one embodiment, the hybridizing of a nucleic acid target, comprising a target nucleic acid sequence, to a nucleic acid probe, comprising a probe nucleic acid sequence is conducted in a hybridization solution comprising a sulfonate buffer.
In a further embodiment, a method for detecting a nucleic acid target is provided, wherein the method includes providing a surface comprising at least 100 nucleic acid probes, each in an area of less than about 0.1 cm2, and each nucleic acid probe having a defined sequence and location on the surface, and contacting the surface with a nucleic acid target, comprising a target nucleic acid sequence, to permit the nucleic acid target to hybridize with at least one selected nucleic acid probe that comprises a probe nucleic acid sequence capable of hybridizing to the target nucleic acid sequence, and wherein the target comprises a binding ligand. The probes are preferably immobilized on the surface, for example by covalent attachment. The method further includes contacting the hydridized target with a receptor comprising multiple sites capable of binding the binding ligand to complex the receptor to the binding ligand; contacting the receptor with an amplification reagent, comprising a plurality of the binding ligands, to complex the amplification reagent to the receptor; and detecting the presence of the complexed amplification reagent. In one embodiment, the amplification reagent comprises an antibody or a DNA matrix, the binding ligand comprises biotin and the receptor comprises streptavidin. In one embodiment, the surface comprising the probes is contacted with the nucleic acid target in a hybridization solution comprising a sulfonate buffer, such as 2-[N-morpholino]ethanesulfonic acid (xe2x80x9cMESxe2x80x9d).
Also provided are complexes including a nucleic acid comprising a binding ligand; a receptor; and an amplification reagent comprising a plurality of the binding ligands. In one embodiment, the binding ligand is biotin and the receptor is streptavidin or avidin.
In another embodiment, there is provided a substrate comprising a surface having immobilized thereon a nucleic acid probe, comprising a probe nucleic acid sequence, hybridized to a nucleic acid target, comprising a target nucleic acid sequence; wherein the target comprises a binding ligand, and wherein the binding ligand on the target is complexed with a receptor comprising multiple sites capable of binding the binding ligand, and wherein the receptor is complexed to an amplification reagent, comprising a plurality of the binding ligands.